Caspase Activation in MCF7 Cells Responding to Etoposide Treatment

Abstract

Studies of the biochemical mechanisms evoked by conventional treatments for neoplastic diseases point to apoptosis as a key process for elimination of unwanted cells. Although the pathways through which chemotherapeutics promote cell death remain largely unknown, caspase proteases play a central role in the induction of apoptosis in response to a variety of stimuli including tumor necrosis factor, fas ligand, and growth factor deprivation. In this article, we demonstrate the induction of caspase protease activity in MCF7 human breast carcinoma cells exposed to the topoisomerase inhibitor, etoposide. Caspase protease activity was assessed by incubating cell lysates with the known caspase substrates,We observed maximal cleavage of acetyl-L-aspartic-L-glutamic-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin within 6 hr following etoposide addition, a time that precedes cell death. In contrast, acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin was resistant to cleavage activity. This substrate cleavage specificity implies that a caspase-3-like protease is activated in response to DNA damage. Consistent with the lysate protease activity, an intracellular marker of caspase activation, poly-ADP ribose polymerase (PARP), was cleaved in a concentration-and time-dependent manner after etoposide-treatment. PARP cleavage followed caspase activation and reached maximum cleavage between 12 and 16 hr. Incubation of the cells with the peptidic caspase inhibitor z-valine-alanine-asparagine-CH 2 F prevented caspase activation, inhibited PARP cleavage, and inhibited cell death. Thus, etoposide killing of MCF7 cells requires a caspase-3-like protease.

Time-course induction of apoptosis by wild-type and attenuated strains of rubella virus

“…The 'cyclic' pattern of caspase activity seen in RV-infected cells resembles that of chemical teratogens (e.g. etoposide), suggesting similar mechanisms of activation of the caspase cascade (Au et al, 1999;Benjamin et al, 1998). However, in this study, assays incorporating the use of apoptotic inhibitor z-VAD-fmk suggest that additional cellular pathways may be involved during RV-induced apoptosis.…”

Lack of p53 induction in fish cells by model chemotherapeutics

“…Cells were treated with the following chemicals and doses: bleomycin (0-100 mg/ml), camptothecin (0-20 mM), cisplatin (0-100 mM), etoposide (0-200 mM), 5-fluorouracil (0-25 mM), and mitomycin C (0-25 mM). Chemical doses were chosen based on concentrations used in published mammalian studies that demonstrated p53 induction and/or apoptosis (Nelson and Kastan, 1994;Benjamin et al, 1998;Houser et al, 2001;Okamura et al, 2004). Dose concentrations were lowered if excessive cell mortality was observed.…”

Influence of Environmental Conditions on c-Jun N-terminal Kinase Mediated Apoptosis of HL60 Cells by Anti-Cancer Drugs

“…Apoptotic pathways after treatment with anti-cancer drugs seemed to be different between HL60/RPMI (daunorubicin and etoposide, caspase 3 dependent, but caspase 8 or 9 in-dependent; vinblastine, caspase 3 independent) and HL60/IMDM (caspase 3 and caspase 9 dependent). The anti-cancer drugs that were used in this study have been known to induce apoptosis, at least in part, through caspase 3 activation (Gamen et al, 1997;Benjamin et al, 1998;Tashiro et al, 1998;Turnbull et al, 1999). In this study, however, we could not find caspase 3 activation after vinblastine treatment in HL60/RPMI cells and expression of apoptotic protein, BID, was consistent with caspase 3 activation.…”